lncRNA-6617调控猪肌内前体脂肪细胞分化的筛选与功能研究

doi:10.11843/j.issn.0366-6964.2022.06.006

Abstract

Abstract: The aim of this study was to investigate the biological characteristics of lncRNA-6617 and its effect on the differentiation of porcine intramuscular preadipocytes, preliminarily explore its upstream regulatory mechanism, and to provide the base for further clarifying the regulation mechanism of intramuscular fat deposition in pigs. In this study, healthy Mashen boars (n=3) and Large White boars (n=3) of 1, 90, and 180 days of age were raised under the same conditions. RNA-seq technology was used to screen muscle-specific lncRNAs in fatty-type pigs (Mashen pigs) and lean-type pigs (Large White pigs). Bioinformatics analysis, RT-PCR and Sanger sequencing were used to identify lncRNA-6617 biological characteristics and expression characteristics in pig tissues. The effect of lncRNA-6617 on the differentiation ability of intramuscular adipocytes were detected after interfering with lncRNA-6617 in porcine intramuscular preadipocytes. Subsequently, the expression differences of m⁶A modification-related enzymes in different breeds of pigs were detected, and the upstream regulatory mechanism of lncRNA-6617 was studied. The results showed that lncRNA-6617 was located on pig chromosome 18, the third intron region of the RAMP3 gene antisense strand, had no protein coding ability, and mainly distributed in the nucleus. lncRNA-6617 was expressed in all detected pig tissues, with the highest expression in back subcutaneous fat, and its expression was significantly higher in MS pigs muscle tissue than that in LW pigs(P < 0.01). In the differentiation process of porcine intramuscular preadipocytes, the expression level of lncRNA-6617 showed an upward trend and reached the peak on day 5. After interfering with lncRNA-6617, the number of differentiated mature adipocytes were significantly reduced, and the expression of key adipogenic genes CEBPα, PPARγ and aP2 were significantly down-regulated (P < 0.01) on day 7 of differentiation. The expression of m⁶A-modified demethylase FTO were significantly higher in the longissimus dorsi and biceps femoris muscle of MS pigs than that of LW pigs (P < 0.01), and were significantly higher than that of LW pigs in the psoas muscle (P < 0.05). And the expression of upstream regulator ZNF217 were significantly higher in the muscle tissue of MS pigs than that in LW pigs (P < 0.01), which was consistent with the expression pattern of lncRNA-6617. After interfering with FTO, the expression of lncRNA-6617 was significantly down-regulated (P < 0.01). In conclusion, lncRNA-6617 highly expressed in the muscle tissue of Mashen pig was screened and identified. The function of lncRNA-6617 in promoting the differentiation of porcine intramuscular preadipocytes and its upstream regulation mechanism were further studied, which riched the epigenetic regulation network of adipogenesis.

Key words: Mashen pig, lncRNA-6617, m⁶A, intramuscular adipocytes, adipogenic differentiation

CLC Number:

S828.2

MENG Shan, YANG Yang, LI Ruixiao, JI Mengting, ZHANG Na, LU Chang, CAI Chunbo, GAO Pengfei, GUO Xiaohong, CAO Guoqing, LI Bugao. Screening and Functional Study of lncRNA-6617 Regulating Porcine Intramuscular Preadipocytes Differentiation[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1712-1722.

References 40

[1]	LIANG W, YE D D. The potential of adipokines as biomarkers and therapeutic agents for vascular complications in type 2 diabetes mellitus[J]. Cytokine Growth Factor Rev, 2019, 48: 32-39.
[2]	BRADLEY D, SHANTARAM D, SMITH A, et al. Adipose tissue T regulatory cells: implications for health and disease[J]. Adv Exp Med Biol, 2021, 1278: 125-139.
[3]	HOCQUETTE J F, GONDRET F, BAÉZA E, et al. Intramuscular fat content in meat-producing animals: development, genetic and nutritional control, and identification of putative markers[J]. Animal, 2010, 4(2): 303-319.
[4]	ZHANG N F, CHENG Z M, LE B Y, et al. Isolation, culture and adipogenic differention of pig myogenic preadipocytes cell[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(12): 2612-2621. (in Chinese)张宁芳, 成志敏, 乐宝玉, 等. 猪肌源性前体脂肪细胞的分离培养和成脂诱导分化研究[J]. 畜牧兽医学报, 2018, 49(12): 2612-2621.
[5]	BERRY D C, STENESEN D, ZEVE D, et al. The developmental origins of adipose tissue[J]. Development, 2013, 140(19): 3939-3949.
[6]	RODRIGUEZ A M, ELABD C, DELTEIL F, et al. Adipocyte differentiation of multipotent cells established from human adipose tissue[J]. Biochem Biophys Res Commun, 2004, 315(2): 255-263.
[7]	YANG Q, WAN Q, ZHANG L T, et al. Analysis of LncRNA expression in cell differentiation[J]. RNA Biol, 2018, 15(3): 413-422.
[8]	REY F, URRATA V, GILARDINI L, et al. Role of long non-coding RNAs in adipogenesis: state of the art and implications in obesity and obesity-associated diseases[J]. Obes Rev, 2021, 22(7): e13203.
[9]	SQUILLARO T, PELUSO G, GALDERISI U, et al. Long non-coding RNAs in regulation of adipogenesis and adipose tissue function[J]. Elife, 2020, 9: e59053.
[10]	GHAFOURI-FARD S, TAHERI M. The expression profile and role of non-coding RNAs in obesity[J]. Eur J Pharmacol, 2021, 892: 173809.
[11]	MEYER K D, SALETORE Y, ZUMBO P, et al. Comprehensive analysis of mRNA methylation reveals enrichment in 3'UTRs and near stop codons[J]. Cell, 2012, 149(7): 1635-1646.
[12]	WARDA A S, KRETSCHMER J, HACKERT P, et al. Human METTL16 is a N6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs[J]. EMBO Rep, 2017, 18(11): 2004-2014.
[13]	LIU H M, XU Y X, YAO B, et al. A novel N6-methyladenosine (m6A)-dependent fate decision for the lncRNA THOR[J]. Cell Death Dis, 2020, 11(8): 613.
[14]	LIU P H, ZHANG B, CHEN Z, et al. m6A-induced lncRNA MALAT1 aggravates renal fibrogenesis in obstructive nephropathy through the miR-145/FAK pathway[J]. Aging (Albany NY), 2020, 12(6): 5280-5299.
[15]	ZHAO X, YANG Y, SUN B F, et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis[J]. Cell Res, 2014, 24(12): 1403-1419.
[16]	GAO P F, CHENG Z M, LI M, et al. Selection of candidate genes affecting meat quality and preliminary exploration of related molecular mechanisms in the Mashen pig[J]. Asian-Australas J Anim Sci, 2019, 32(8): 1084-1094.
[17]	GUO X H, QIN B Y, YANG X F, et al. Comparison of carcass traits, meat quality and expressions of MyHCs in muscles between Mashen and Large White pigs[J]. Ital J Anim Sci, 2019, 18(1): 1410-1418.
[18]	ZHAO J X, LI K, YANG Q Y, et al. Enhanced adipogenesis in Mashen pigs compared with Large White pigs[J]. Ital J Anim Sci, 2017, 16(2): 217-225.
[19]	GAO P F, GUO X H, DU M, et al. LncRNA profiling of skeletal muscles in Large White pigs and Mashen pigs during development, [J]. J Anim Sci, 2017, 95(10): 4239-4250.
[20]	ROBINSON M D, MCCARTHY D J, SMYTH G K. edgeR: a bioconductor package for differential expression analysis of digital gene expression data[J]. Bioinformatics, 2010, 26(1): 139-140.
[21]	GHOSH S, CHAN C K K. Analysis of RNA-Seq data using TopHat and cufflinks[J]. Methods Mol Biol, 2016, 1374: 339-361.
[22]	KONG L, ZHANG Y, YE Z Q, et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine[J]. Nucleic Acids Res, 2007, 35(S2): W345-W349.
[23]	WANG L G, PARK H J, DASARI S, et al. CPAT: coding-potential assessment tool using an alignment-free logistic regression model[J]. Nucleic Acids Res, 2013, 41(6): e74.
[24]	RUIZ-OJEDA F J, RUPÉREZ A I, GOMEZ-LLORENTE C, et al. Cell models and their application for studying adipogenic differentiation in relation to obesity: a review[J]. Int J Mol Sci, 2016, 17(7): 1040.
[25]	SUN L, GOFF L A, TRAPNELL C, et al. Long noncoding RNAs regulate adipogenesis[J]. Proc Natl Acad Sci U S A, 2013, 110(9): 3387-3392.
[26]	JANDURA A, KRAUSE H M. The new RNA world: growing evidence for long noncoding RNA functionality[J]. Trends Genet, 2017, 33(10): 665-676.
[27]	STATELLO L, GUO C J, CHEN L L, et al. Gene regulation by long non-coding RNAs and its biological functions[J]. Nat Rev Mol Cell Biol, 2021, 22(2): 96-118.
[28]	ZHU E D, ZHANG J J, LI Y C, et al. Long noncoding RNA Plnc1 controls adipocyte differentiation by regulating peroxisome proliferator-activated receptor γ[J]. FASEB J, 2019, 33(2): 2396-2408.
[29]	ZUO C Q, PAN Y Q, LENG D, et al. Transcriptome analysis of long non-coding RNAs reveals NR_015556 lncRNA is a novel regulator for adipocyte differentiation[J]. Biochem Biophys Res Commun, 2022, 601: 79-85.
[30]	CHEN C, CUI Q M, ZHANG X, et al. Long non-coding RNAs regulation in adipogenesis and lipid metabolism: emerging insights in obesity[J]. Cell Signal, 2018, 51: 47-58.
[31]	SUN Y M, CAI R, WANG Y Q, et al. A newly identified LncRNA LncIMF4 controls adipogenesis of porcine intramuscular preadipocyte through attenuating autophagy to inhibit lipolysis[J]. Animals (Basel), 2020, 10(6): 926.
[32]	WANG J, CHEN M Y, CHEN J F, et al. LncRNA IMFlnc1 promotes porcine intramuscular adipocyte adipogenesis by sponging miR-199a-5p to up-regulate CAV-1[J]. BMC Mol Cell Biol, 2020, 21(1): 77.
[33]	LIU Y B, AO H, XING K, et al. Research progress on fat-related lncRNAs in animals[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(7): 2021-2029. (in Chinese)刘怡冰, 敖红, 邢凯, 等. 脂肪相关lncRNAs在动物中的研究进展[J]. 中国畜牧兽医, 2019, 46(7): 2021-2029.
[34]	DU C, FU S Y, GAO H Y, et al. Transcriptome analysis of intramuscular preadipocytes and matureadipocyte in cashmere goats[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(5): 714-721. (in Chinese)杜琛, 付绍印, 高鸿雁, 等. 绒山羊肌内脂肪细胞成熟前后比较转录组分析[J]. 畜牧兽医学报, 2014, 45(5): 714-721.
[35]	XIE G J, WANG Y, XU Q, et al. Selection and validation of the differentially expressed genes during the adipogenic differentiation of Jianzhou Da'er goat intramuscular adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(7): 1525-1536. (in Chinese)谢光杰, 王永, 许晴, 等. 简州大耳羊肌内脂肪细胞成脂分化差异表达基因的筛选与鉴定[J]. 畜牧兽医学报, 2020, 51(7): 1525-1536.
[36]	LI A, ZHANG X X, HUANG W L, et al. Identification and analysis of circRNAs in intramuscular adipose tissues between Large White and Laiwu pigs[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(7): 1343-1353. (in Chinese)李嫒, 张秀秀, 黄万龙, 等. 大白猪和莱芜猪肌内脂肪组织circRNAs的鉴定与分析[J]. 畜牧兽医学报, 2018, 49(7): 1343-1353.
[37]	CHI K R. The RNA code comes into focus[J]. Nature, 2017, 542(7642): 503-506.
[38]	ZHU L N. Effect of FTO, METTL3 gene expression on mRNA m6A methylation and lipid metabolism in porcine subcutaneous adipocytes[D]. Hangzhou: Zhejiang University, 2014. (in Chinese)朱琳娜. FTO、METTL3基因表达对猪脂肪细胞mRNA N6-甲基腺苷水平及脂肪沉积的影响研究[D]. 杭州: 浙江大学, 2014.
[39]	JIANG Q. Effects and mechanisms of key mRNA m6A genes in porcine intramuscular fat deposition[D]. Hangzhou: Zhejiang University, 2018. (in Chinese)江芹. mRNA m6A修饰关键基因对猪肌内脂肪沉积的影响及机制[D]. 杭州: 浙江大学, 2018.
[40]	WANG X X, ZHU L N, CHEN J Q, et al. mRNA m6A methylation downregulates adipogenesis in porcine adipocytes[J]. Biochem Biophys Res Commun, 2015, 459(2): 201-207.

Screening and Functional Study of lncRNA-6617 Regulating Porcine Intramuscular Preadipocytes Differentiation

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References 40

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	HAN Haozhe, TIE Zihang, PANG Weijun, CAI Rui. Advances of IGF2BP2-Mediated m⁶A Modification on Animal Fat Deposition [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3605-3612.
[2]	QUBI Wuqie, LI Yanyan, LI Xin, WANG Yong, WANG Youli, LIU Wei, ZHU Jiangjiang, LIN Yaqiu. APOA4 Gene Inhibits Intramuscular Adipocyte Differentiation in Goat [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 1927-1938.
[3]	YANG Zhimei, LIANG Chengcheng, ZHANG Dianqi, LI Xuefeng, ZAN Linsen. Research Progress on the Regulation of circRNA by m⁶A Modification [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4016-4027.
[4]	SHI Mingyue, ZHANG Xuelian, YANG Xiaofen, NIU Jin, XING Jiandong, LU Chang, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Study on NR1H3 Gene Regulating Differentiation of Porcine Preadipocyte [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2094-2103.
[5]	ZHANG Hao, WANG Yong, LI Yanyan, LUO Cheng, LI Xin, LI Zhixiong, ZHU Jiangjiang, LIN Yaqiu. Bioinformatics Analysis of Goat RPL26 Gene and Its Effect on Intramuscular Adipocyte Differentiation [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1064-1076.
[6]	Relationship between Porcine m, GAO Zhongcheng, BAO Wenbin, WU Zhengchang, WU Shenglong. Relationship between Porcine m⁶A Methylase WTAP Gene and F18 Escherichia coli Infection [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1709-1716.
[7]	CAI Chunbo, LIU Min, YANG Yang, ZHANG Wanfeng, GAO Pengfei, CAO Guoqing, LI Bugao, GUO Xiaohong. Regulation of miR-186-5p on Proliferation and Differentiation of Porcine Primary Preadipocytes [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1247-1257.
[8]	CAI Chunbo, ZHANG Xuelian, ZHANG Wanfeng, YANG Yang, GAO Pengfei, GUO Xiaohong, LI Bugao, CAO Guoqing. Evaluation of Genetic Structure in Mashen Pigs Conserved Population Based on SNP Chip [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(4): 920-931.
[9]	LIANG Xiaojuan, TAO Cong, ZHAO Ying, WANG Chao, LIU Lulu, WANG Yanfang. Effect of RNF20 and It-mediated Monoubiquitination of Histone H2B at Lysine 120 (H2Bub) on Brown Adipocyte Differentiation in Mice [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 722-731.
[10]	CUI Sheng, LIN Yaqiu, XU Qing, ZHU Jiangjiang, WANG Yong. Interfering Smad3 Promotes Goat Adipocyte Differentiation [J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(3): 475-489.
[11]	ZHANG Kaiyi, ZHU Wenjuan, XIE Ning, YE Huaqiong, YANG Shulin. Establishment of a Spontaneous Insulin Resistant 3T3-L1 Adipocyte Cell Model [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(7): 1475-1485.
[12]	REN Ling, HU Xin, XING Yishen, WANG Yahui, LI Junya, ZHANG Lupei. Effects of S100a10 on White and Brown Adipogenic Differentiation of Mice Preadipocytes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(6): 1171-1178.
[13]	LU Zengkui, ZHANG Liping, LI Qing, LIU Enmin, DU Lixin, CHU Mingxing, WEI Caihong. N⁶-methyladenosine Modification in mRNA and Its Research Advance in Animals [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(1): 1-13.
[14]	LIN Sen, LIN Ya-qiu, ZHU Jiang-jiang, XU Qing, ZHAO Yue, CHI Yong-dong, WANG Yong. Effect of Wnt10b on the Expression of Precursor Adipocytes Differentiation Related Genes in Goat [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(4): 685-692.
[15]	ZHANG Ning-fang, CHENG Zhi-min, LE Bao-yu, QIN Ben-yuan, WANG Yuan-yuan, WANG He-jie, GAO Peng-fei, GUO Xiao-hong, LI Bu-gao, CAO Guo-qing. Isolation, Culture and Adipogenic Differention of Pig Myogenic Preadipocytes Cell [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(12): 2612-2621.